Overall numbers of T cells remain constant despite input of new T cells from the thymus and antigenic challenges that result in increases in the numbers of specific T cell subsets. This T cell homeostasis is mediated by controls on T cell proliferation and death. Activated T cell death is promoted by multiple factors including withdrawal of cytokines and migration of death receptors. We have found that the cytokine IL-6 can promote survival of resting but not activated T cells. This led us to hypothesize that T cell survival depends not only on cytokine levels but also on activation-induced changes in cytokine receptor signaling. Consistent with this motion, we observed a loss of IL-6-mediated STAT1 phosphorylation in activated T cells. We now propose to determine whether the loss of IL-6-mediated STAT1 phosphorylation is responsible for the failure of IL-6 to rescue activated T cells from apoptosis (Aim I). To do this we will use SH2 domain swapping techniques and site-directed mutagenesis to alter STAT1 and the IL-6 receptor subunit, gp130, in ways that will allow IL-6 to induce STAT1 phosphorylation in activated T cells. These cells will be tested for their ability to respond to IL-6 in survival assays. We will also investigate the mechanism of the activation- induced inhibition of IL-6 signaling (Aim II). Most likely, either the STAT1 binding site on gp130 is not phosphorylated or the Jak kinases which are responsible for STAT1 phosphorylation are not activated. We will examine the phosphorylation status of specific tyrosines on gp130 by phosphoamino acid analysis and the activation status of the Jak kinases by immunoblotting and kinase assays. We also propose to use cDNA microarray technology to investigate the consequences of the loss of STAT1 phosphorylation on the transcription of genes involved in cell survival and differentiation (Aim III). Finally, we will engineer a transgenic mouse in which IL-6-mediated STAT1 phosphorylation is preserved in activated T cells and examine the consequences for T cell homeostasis and autoimmunity (Aim IV). The discovery and characterization of the inhibitory mechanism responsible for the deficiency in IL-6-mediated STAT1 phosphorylation should enrich our understanding of the role of IL-6 in T cell homeostasis and differentiation.